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. 2015 Apr 13;209(1):85-95.
doi: 10.1083/jcb.201409064.

Sac2/INPP5F Is an Inositol 4-phosphatase That Functions in the Endocytic Pathway

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Free PMC article

Sac2/INPP5F Is an Inositol 4-phosphatase That Functions in the Endocytic Pathway

Fubito Nakatsu et al. J Cell Biol. .
Free PMC article

Abstract

The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL-Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.

Figures

Figure 1.
Figure 1.
Localization of Sac2 on endosomes. (A and B) GFP- or mCherry (mCh)-Sac2 is present on Rab5-positive endosomes, as shown by colocalization with endogenous Rab5 immunoreactivity (A) or with GFP-Rab5 (B). (C and D) GFP-Sac2 also colocalizes with the majority of APPL1 (RFP-APPL1)-positive endosomes (C), but does not colocalize with a lysosomal marker protein, RFP-LAMP1 (D). In A, B, and C, examples of colocalizations are indicated by arrows. Images were taken by spinning-disc confocal microscopy. Bars, 5 µm.
Figure 2.
Figure 2.
Colocalization of Sac2 with OCRL on early endosomes and at the last stage of clathrin-mediated endocytosis. Live spinning-disc confocal microscopy. (A) Colocalization of GFP-Sac2 and mCherry-OCRL. The majority of the fluorescent puncta positive for both proteins are expected to be early endosomes, given the close colocalization of both proteins with Rab5 (see Fig. 1 A and Hyvola et al., 2006; Erdmann et al., 2007). Insets show enlarged views of the regions boxed in yellow. (B and C) Time course of CLC-mRFP and either GFP-Sac2 (B) or GFP-OCRL (C) fluorescence intensities at very late stage clathrin-coated pits demonstrating that Sac2 and OCRL appear when clathrin is disappearing, and that Sac2 and OCRL display similar recruitment patterns. The disappearance of the Sac2 and OCRL fluorescence reflects the newly formed vesicle moving out of the focal plane. Bars: (A) 5 µm; (B and C) 1 µm.
Figure 3.
Figure 3.
Recruitment of Sac2 to macropinosomes. Mouse fibroblasts expressing GFP-Sac2 and either RFP-PHAKT (A) or RFP-EEA1 (B) were also cotransfected with H-RasV12G to induce the formation of macropinosomes. The gallery of confocal images shows that Sac2 is recruited to these vesicles when the AKT signal, which primarily reflects PI(3,4,5)P3, is disappearing, and remains associated with the vesicles as they mature to EEA1-positive endosomes. Bars, 1 µm.
Figure 4.
Figure 4.
Defect in the internalization of transferrin in Sac2 KO MEFs. Internalization of biotinylated transferrin over time was analyzed biochemically in WT and Sac2 KO MEFs using an ELISA-based assay (see Materials and methods for details). Data were expressed as mean ± SD (error bars) of three independent experiments (Student’s t test, **, P < 0.01; ***, P < 0.001).
Figure 5.
Figure 5.
Sac2 functions downstream of Rab5 and is part of a complex also comprising OCRL. (A–C) COS7 cells were cotransfected with mCherry (mCh)-Sac2 together with GFP-Rab5WT, GFP-Rab5Q79L (constitutively active; B), or GFP-Rab5S34N (dominant negative; C), as indicated, and imaged by confocal microscopy. mCh-Sac2 colocalizes with WT and constitutively active Rab5. In contrast, it has a predominant cytosolic localization in cells expressing dominant-negative Rab5. Inset panels show enlarged views of the regions boxed in yellow. Bars: (full-size images) 5 µm; (insets) 2 µm for inset image. (D) COS7 cells were cotransfected with 3×Flag-Sac2, HA-OCRL, and GFP-tagged Rab5 constructs (Rab5WT, GFP-Rab5Q79L, or GFP-Rab5S34N). Cell lysates were immunoprecipitated with anti-Flag antibody to enrich for Sac2. Immunoblotting of the starting lysates (left) and of the immunoprecipitates (right) for the Flag, HA, and GFP epitopes confirms enrichment of Sac2 and shows robust coprecipitation of Rab5 and OCRL only from cells expressing GFP-Rab5WT or GFP-Rab5Q79L. Recovery of OCRL and Rab5 was dramatically reduced from cells expressing Rab5S34N.
Figure 6.
Figure 6.
Sac2 is an inositol 4-phosphatase. (A) GFP, GFP-Sac2, and GFP-Sac2D460N were transiently overexpressed in Expi293 HEK cells and semi-purified by immunoprecipitation using anti-GFP antibody. The presence of inositol-phosphatase activity in the immunoprecipitated material was assessed with a malachite-based assay. Data shown represent mean ± SD (error bars). (B) Lysates of COS-7 cells expressing GFP, GFP-Sac2WT, or GFP-Sac2D460N were subjected to anti-GFP immunoprecipitation, and immunoprecipitates were processed by immunoblotting to assess the enrichment of GFP and GFP fusion proteins (bottom), of inositol phosphatases (OCRL, INPP5B, and synaptojanin 1), and of control proteins (epsin 1 and Erk1/2). Note the enrichment of OCRL and INPP5B in anti-GFP immunoprecipates from COS7 cells expressing GFP-Sac2WT or GFP-Sac2D460N. (C) GST-SacWT and GST-SacD460N were expressed in bacteria, purified on glutathione Sepharose beads, and analyzed for inositol phosphatase activity against the seven PIs via the malachite assay. Data shown represent mean ± SD (error bars).

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